A Step Toward Regenerating Amputated Limbs

Wearable device helps frogs regrow amputated limbs.

by Dan Ferber

January 22, 2019

Once a limb is amputated, it’s gone forever, and the person has to live the rest of their life as an amputee. At least that’s the way it’s always been. But there is now evidence that a single day wearing a powerful new wearable device causes amputee frogs to partially regrow an amputated hind leg over the next nine months.

The work could lead to new treatments to regrow amputated fingers, arms, legs, and ultimately diseased heart, lung, liver, eyes, and other organs in humans.

To regenerate tissue and organs, engineers typically use a porous biocompatible, biodegradable scaffold with the appropriate shape and mechanical properties. They seed that scaffold with one or more types of lab-grown cells, and often add growth factors, such as molecules that promote tissue growth. When the approach works, cells knit together into a functional tissue or organ that can be implanted to replace diseased or damaged tissue.

The approach can work for simple tissues. But it’s a “complete nonstarter” for growing complex structures like a limb or a hand, says biologist Michael Levin, director of the Allen Discovery Center at Tufts University and senior author on the study.

Levin and his colleagues take a different approach entirely. Since our body forms during embryonic development, Levin believes “There’s got to be a [biological] signal that says, ‘Build a limb.” In fact, there must be a complete biological program that steers the development of organs, hands, limbs, and other complex structures, said Levin, who also trained in computer science.

Levin likens these biological programs to subroutines—specific sections of code in a program that carry out a specific task.

While such subroutines are routinely active in embryonic animals, they shut down in adults of many species, which prevents them from regenerating lost body parts. But adults of some species retain some ability to regenerate. Adult crabs can grow new claws, starfish grow new arms, and flatworms snipped in two can grow entirely new individuals. Even deer regrow new antlers each year.

Levin and postdoc Celia Herrera-Rincon suspected that under the right circumstances, those biological programs could be activated in adults. To find the signal or combination of signals that leads to limb regeneration, the two teamed up with biomedical engineer David Kaplan, director of Tufts’ Initiative for Neural Science, Disease and Engineering.

Kaplan’s team had developed biocompatible hydrogels for tissue engineering that are made from silk proteins. A few years ago, his team also engineered a simple device containing a wearable cylinder lined with the silk hydrogel. The idea was to imbue drug candidates, growth factors, or other molecules into the hydrogel, assemble the device, then fasten the device to the site of amputation. The dissolved drug or growth factor would leach into the tissue, helping limbs to regenerate.

The researchers chose to work on African clawed frogs, which biologists have used for decades to study embryonic development. Tadpoles of this species can regenerate tails, but if an adult frog has a leg amputated, they naturally regrow only a cartilage-heavy spike following amputation.

In the recent study, the researchers anesthetized the frogs and amputated their right hind leg, attached the device for 24 hours, then removed it. For some of the frogs, the device had silk hydrogel imbued with progesterone; others were exposed to silk hydrogel alone. Best known for helping steer the human menstrual cycle and early stages of pregnancy, progesterone is also known to helps repair nerves, heal wounds, grow new blood vessels, and reshape bone.

Herrera-Rincon then observed the frogs for nine months. Using digital images, x-rays, and specialized image analysis software to quantitate the results, she and her colleagues showed that frogs treated with progesterone regrew paddle-like structures over nine months, but frogs treated the same way but with no progesterone instead developed cartilage-y spikes. They also swam around a tank faster than animals that received a sham treatment.

Complex, patterned structures, including bone and blood vessels, regrew in progesterone-treated animals but not in untreated animals. Nerves also regrew better into the new limb in these frogs. The treatment also induced changes in the frog’s immune system that promoted healing.

Next, the researchers will mix and match different drugs into drug cocktails. They will test them on frog amputees and choose those that best induce regeneration. If the frog experiments work, the researchers will see if they can induce finger and limb regeneration in mice, whose biology more closely resembles humans, Levin said.

Regeneration drugs tested this way may not be safe, so they’ll have to be thoroughly tested to make sure they don’t disrupt other essential body functions, such as the heartbeat, Levin says. Plus, bioreactors will be modified to test drug cocktails on other animals.

“I think the study was well conducted,” said Cato T. Laurencin, director of the Connecticut Convergence Institute for Regenerative Engineering at the University of Connecticut Health Center in Farmington. “It’s important to study animals that can regenerate to unlock clues to uncover the regenerative process.”

The work is still in its early days, but if regeneration therapies work in animal trials, researchers will test them clinical trials. If successful, those trials could be good news for the more than 2 million amputees in the United States, millions more around the world, and other patients that have had other tissues or organs removed.

“Ultimately, we could regenerate fingers, limbs, and any diseased organs, including heart and lung and liver and eyes,” Levin said. “The sky’s the limit.”